Abstract Oxygen therapy is a common clinical necessity, but it comes with significant negative side effects. Specifically, the hyperoxic condition it produces generates a number of reactive oxygen species, including superoxide anions that cause mitochondrial dysfunction. Although this toxicity is a key factor in the application of oxygen therapy, little is known about how hyperoxia impacts mitochondrial energy production, or the protein regeneration mechanisms that can offer protection from its effects. Our research program focuses on thioredoxin (Trx), a cytoplasmic redox protein that can reduce oxidative stress and regenerate enzymes that oxidation has inactivated. Recently we published that increased expression of Thioredoxin protects the lung injury and increased survival of Trx-Tg mice in hyperoxia, but mice with lower expression of Trx were more sensitive to hyperoxia and suffered significant mortality. However, the mechanism by which high levels of Thioredoxin protects against lung injury remains unknown. Our preliminary data establish that cytoplasmic Trx1 translocates to mitochondria during hyperoxia, but this movement does not occur in dnTrx-Tg mice. These findings propel us to hypothesize that Trx protects mitochondria from hyperoxia by reducing oxidative stress through UCP-2-dependent uncoupling, and furthermore by protecting mitochondrial dysfunction in hyperoxia as superoxide anion generation in the mitochondria is a key mechanism of pulmonary oxygen toxicity. Accordingly, in Aim 1 we will determine whether and how translocated cytoplasmic Trx1 protects against mitochondrial dysfunction in hyperoxia. In Aim 2 we will find if high levels of Trx prevents dynamin- related protein (Drp1) activation and thereby protects against mitochondrial fragmentation and dysfunction. In Aim 3 we will determine if increased translocation of PGC-1? to the nucleus in Trx-Tg mice can protect against the mitochondrial dysfunction caused by hyperoxia. Using state-of-the-art techniques that include mitochondrial flux analysis, EPR spectroscopy, biochemical enzymatic assay, and cutting-edge molecular approaches, we will dissect the role Trx1 plays in protecting the dysfunctional mitochondria in hyperoxia. We will also create a novel conditional Trx knockout mouse, a PGC1a-knockout mouse with increased or decreased expression of Trx to understand in vivo role of high levels of Trx on mitochondrial dysfunction in hyperoxia. The project is expected to provide a clear understanding of the way cytosolic Trx1 affects mitochondrial function during normoxia and hyperoxia. Using the transgenic mice (and cells derived from them) for in vivo and in vitro mechanistic studies, we expect to uncover mitochondrial mechanisms that are modulated by Trx1 during hyperoxia. We believe results produced by the project will incite novel intervention strategies to protect patients against pulmonary toxicity resulting from oxygen therapy.